Florian Gessler

Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation, migration and invasion of malignant glioma cells

Tissue factor (TF) is upregulated in several malignant diseases, including gliomas. Here, we demonstrate pronounced differences in the expression of TF and its interactors factor VII and protease-activated receptor 2 (PAR-2) in nine human glioma cell lines (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304, Hs 683) as detected by RT-PCR and Western blot analysis. Inhibition of TF signaling by a neutralizing monoclonal antibody (mAb TF9-10H10) led to significantly reduced proliferation in high-grade astroglial (MZ-18 and MZ-304) and oligodendroglial (Hs 683) cell lines abundantly expressing TF, but not in U373 cells expressing low amounts of TF. Scratch migration assays and Boyden chamber assays indicated that mAb TF9-10H10 and lentiviral knockdown of TF significantly reduced cell migration and invasion of MZ-18, MZ-304 and Hs 683 cells, both under normoxic and hypoxic conditions. Of note, all three cell lines displayed increased cell migration and invasion under hypoxic conditions (1% O(2)), which was associated with enhanced expression of TF and increased phosphorylation of p44/42 mitogen-activated protein kinase (ERK1/2). Silencing of TF blocked activation of the ERK pathway, induction of TF expression and the potentiating effect of hypoxia on cell migration and invasion. RNA interference against PAR-2 abrogated the autocrine effects of TF on cell proliferation, migration and invasion, indicating that TF signals via PAR-2 in glioma cells. Our results suggest an important role for the TF/FVIIa/PAR-2/ERK axis in tumor growth and invasion of glioma and suggest that TF may be a suitable target for the development of novel therapies against high-grade glioma.

Chemoresistance is associated with increased cytoprotective autophagy and diminished apoptosis in bladder cancer cells treated with the BH3 mimetic ()-Gossypol (AT-101)

BackgroundAcquired resistance to standard chemotherapy causes treatment failure in patients with metastatic bladder cancer. Overexpression of pro-survival Bcl-2 family proteins has been associated with a poor chemotherapeutic response, suggesting that Bcl-2-targeted therapy may be a feasible strategy in patients with these tumors. The small-molecule pan-Bcl-2 inhibitor (−)-gossypol (AT-101) is known to induce apoptotic cell death, but can also induce autophagy through release of the pro-autophagic BH3 only protein Beclin-1 from Bcl-2. The potential therapeutic effects of (−)-gossypol in chemoresistant bladder cancer and the role of autophagy in this context are hitherto unknown.MethodsCisplatin (5637rCDDP1000, RT4rCDDP1000) and gemcitabine (5637rGEMCI20, RT4rGEMCI20) chemoresistant sub-lines of the chemo-sensitive bladder cancer cell lines 5637 and RT4 were established for the investigation of acquired resistance mechanisms. Cell lines carrying a stable lentiviral knockdown of the core autophagy regulator ATG5 were created from chemosensitive 5637 and chemoresistant 5637rGEMCI20 and 5637rCDDP1000 cell lines. Cell death and autophagy were quantified by FACS analysis of propidium iodide, Annexin and Lysotracker staining, as well as LC3 translocation.ResultsHere we demonstrate that (−)-gossypol induces an apoptotic type of cell death in 5637 and RT4 cells which is partially inhibited by the pan-caspase inhibitor z-VAD. Cisplatin- and gemcitabine-resistant bladder cancer cells exhibit enhanced basal and drug-induced autophagosome formation and lysosomal activity which is accompanied by an attenuated apoptotic cell death after treatment with both (−)-gossypol and ABT-737, a Bcl-2 inhibitor which spares Mcl-1, in comparison to parental cells. Knockdown of ATG5 and inhibition of autophagy by 3-MA had no discernible effect on apoptotic cell death induced by (−)-gossypol and ABT-737 in parental 5637 cells, but evoked a significant increase in early apoptosis and overall cell death in BH3 mimetic-treated 5637rGEMCI20 and 5637rCDDP1000 cells.ConclusionsOur findings show for the first time that (−)-gossypol concomitantly triggers apoptosis and a cytoprotective type of autophagy in bladder cancer and support the notion that enhanced autophagy may underlie the chemoresistant phenotype of these tumors. Simultaneous targeting of Bcl-2 proteins and the autophagy pathway may be an efficient new strategy to overcome their “autophagy addiction” and acquired resistance to current therapy.

Knockdown of TFPI-2 promotes migration and invasion of glioma cells

Glioblastoma is the most malignant primary brain tumor. Due to its highly promigratory and proinvasive properties, standard therapy including surgery, chemotherapy and radiation fails in eradicating this highly aggressive type of cancer. Here, we evaluated the role of TFPI-2, a Kunitz-type serine protease inhibitor, which has been previously described as a tumor suppressor gene in several types of cancer, including glioma. TFPI-2 expression was absent in five of nine investigated high-grade glioma cell lines. Lentiviral knockdown of TFPI-2 in two of the TFPI-2-expressing cell lines (MZ-18 and Hs 638) was associated with pronounced changes in the cellular behavior: glioma cell proliferation, migration and invasion were significantly increased in TFPI-2 knockdown cells in comparison to empty vector-transfected control cells. Since TFPI-2 might exert its tumor suppressor function by inhibiting MMPs, we subsequently analyzed the effects of specific MMP inhibitors on cell invasion of TFPI-2 KD cells vs. control cells. The data obtained from these experiments suggest that the anti-invasive properties of TFPI-2 are associated with inhibition of MMP-1 and MMP-2, while inhibition of MMP-9 seems to play a minor role in this context. Our findings underscore the important role of TFPI-2 as a tumor suppressor gene and indicate that TFPI-2 may be a useful diagnostic marker for the aggressive phenotype of glial tumors.

Combination of Intraoperative Magnetic Resonance Imaging and Intraoperative Fluorescence to Enhance the Resection of Contrast Enhancing Gliomas.

BACKGROUND Evidence suggests that extent of resection (EOR) is a prognostic factor for patients harboring gliomas. Recent studies have displayed the importance of intraoperative magnetic resonance imaging (iMRI) with 5-aminolevulinic acid (5-ALA) fluorescence-guidance in order to maximize EOR. OBJECTIVE To compare iMRI and 5-ALA fluorescence-guidance and the impact on patient survival. METHODS Thirty-two patients with contrast-enhancing gliomas undergoing intended gross total resection (GTR) were included in a prospective study. Surgeries were started under white-light conditions. When GTR was thought to be achieved, an iMRI scan was performed and a blue light turned on to search for unintentionally remaining tumor tissue. iMRI findings were compared with intraoperative fluorescence findings. Histological examination of tumor bulk and any additionally resected tissue was performed. All patients underwent early postoperative high-field MRI to determine EOR. RESULTS In 13 patients (40.6%), iMRI and fluorescence unequivocally did not show residual tumor intraoperatively. In 19 patients (59.4%), resection was continued due to iMRI or fluorescence findings. In 9 of these (47.4%), iMRI and fluorescence findings were inconsistent regarding residual tumor. GTR according to postoperative MRI was achieved in all but 1 patient. Histological examination ruled out false positive findings in all additionally resected specimens. Sensitivity and specificity to detect residual tumor tissue were 75% and 100%, respectively, for iMRI and 70% and 100% for 5-ALA fluorescence. CONCLUSION Use of iMRI as well as fluorescence-guidance are appropriate methods to improve the extent of resection in surgery of contrast-enhancing gliomas. Best results can be achieved by complementary use of both modalities.

Anti-tissue factor (TF9-10H10) treatment reduces tumor cell invasiveness in a novel migratory glioma model

In vitro and descriptive studies of human tissue samples revealed the pro‐coagulant glycoprotein tissue factor (TF) as a potent player in glioma cell infiltration that is activated by hypoxia and has also been shown to be upregulated by mutations of TP53 or PTEN. Here we present the morphological and genetic characterization of a novel glioblastoma in vivo model and provide evidence that treatment with an antibody targeting TF leads to reduced glioma cell invasiveness. Therefore, we established a murine xenograft treatment model by transplanting the angiogenic and diffusely infiltrating human glioma cell line MZ‐18 with endogenous TF expression into nude mice brains and treating these mice with an intracranial osmotic pump system continuously infusing a monoclonal antibody against TF (mAb TF9‐10H10). The human MZ‐18 cell line harbors two TP53 mutations resulting in a strong nuclear accumulation of p53, thereby facilitating the unambiguous identification of tumor cells in the xenograft model. Intracranial application of TF9‐10H10 significantly reduced invasion of MZ‐18 cells compared to mock‐treated control animals. The extent of activated blood vessels was also reduced upon anti‐TF treatment. Thus, targeting the TF pathway might be a promising treatment strategy for future glioblastoma therapies, by affecting both invading tumor cells and tumor vasculature.

Interference with the HSF1/HSP70/BAG3 pathway primes glioma cells to matrix detachment and BH3 mimetic-induced apoptosis

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101–induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell–matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156–68. ©2016 AACR.

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumours. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM) which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic-ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this point of view article we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.

The Impact of Tracheostomy Timing on Clinical Outcome and Adverse Events in Poor-Grade Subarachnoid Hemorrhage.

Objective: The value of optimal timing of tracheostomy in patients with subarachnoid hemorrhage is controversially debated. This study investigates whether early or late tracheostomy is associated with beneficial outcome or reduced rates of adverse events. Design: Retrospective observational multicentric on patients prospectively inserted into a database. Setting: Neurologic ICUs of one academic hospital and two secondary hospitals in Germany. Patients: Data of all patients admitted to the Goethe University Hospital between 2006 and 2011 with poor-grade subarachnoid hemorrhage were prospectively entered into a database. All patients who underwent tracheostomy were included for analysis. Follow-up was maintained in primary and secondary ICUs. Interventions: Patients underwent tracheostomy upon expected long-term ventilation. Early tracheostomy was defined as performed on days 1–7 and late tracheostomy on days 8–20 after admission. Measurement and Main Results: We compared 148 consecutive patients admitted with poor-grade (World Federation of Neurosurgical Societies, 3–5) subarachnoid hemorrhage. Early tracheostomy was performed in 39 patients and late tracheostomy in 109 patients. In early versus late tracheostomy groups, no significant differences were observed with regard to ICU mortality (7.7% vs 7.3%; p = 0.93) and median modified Rankin Scale after 6 months (3 vs 3; p = 0.94). Of the early group, pneumonia developed in 19 patients, whereas in the late group, pneumonia developed in 75 patients (48.7% vs 68.8%; p = 0.03; odds ratio, 2.32; 95% CI, 1.1–4.9). Six patients of the early group (15.4%) and 36 patients of the late group (33%) suffered from respiratory adverse event (p = 0.04; odds ratio, 2.71; 95% CI, 1.04–7.06). Mechanical ventilation was shorter (17.4 vs 22.3 d; p < 0.05) and decannulation occurred earlier (42 vs 54 d; p = 0.039) in the early tracheostomy group. Conclusions: Tracheostomy within 7 days of critical care admission is a feasible and safe procedure for patients with poor-grade subarachnoid hemorrhage. Early tracheostomy was not associated with an improvement in mortality or neurologic outcome but associated with fewer respiratory adverse events.

Neural stem cell transplantation in ischemic stroke: A role for preconditioning and cellular engineering:

Ischemic stroke continues to be a leading cause of morbidity and mortality throughout the world. To protect and/or repair the ischemic brain, a multitiered approach may be centered on neural stem cell (NSC) transplantation. Transplanted NSCs exert beneficial effects not only via structural replacement, but also via immunomodulatory and/or neurotrophic actions. Unfortunately, the clinical translation of such promising therapies remains elusive, in part due to their limited persistence/survivability within the hostile ischemic microenvironment. Herein, we discuss current approaches for the development of NSCs more amenable to survival within the ischemic brain as a tool for future cellular therapies in stroke.

AT-101 simultaneously triggers apoptosis and a cytoprotective type of autophagy irrespective of expression levels and the subcellular localization of Bcl-xL and Bcl-2 in MCF7 cells

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.